Tissue-type plasminogen activator (tPA) belongs to the chymotrypsin family of serine proteases and catalyzes the rate-limiting step in the endogenous fibrinolytic cascade, which converts circulating zymogen plasminogen into active plasmin. Plasmin in turn lyses fibrin by hydrolyzing peptide bonds in the triple-stranded connector rod regions. Five distinct structural domains make up the active human tPA protein. The DNA and amino acid sequences of human tPA were described by Pennica et al. (Nature 301:214-221, 1983).
tPA is synthesized as a single-chain polypeptide of approximately 72 kDa and converted into an active two-chain form by proteolytic cleavage at residues 275-276. This cleavage is accompanied by an increase in fibrinolytic activity. The two-chain form consists of: a 33 kDa light chain derived from the C-terminus with complete catalytic activity (C); and a 38 kDa heavy chain derived from the N-terminus with no catalytic activity. The N-terminal portion of the molecule contains four distinct structural domains including a fibronectin-like finger domain (F), an epidermal growth factor (EGF) homologous region, and two kringle structures (K1 and K2). The C-terminal portion contains what is known as the serine protease (SP) domain and comprises the active site for the fibrin-specific serine protease activity. tPA's role in fibrinolysis and potential as a therapeutic candidate for treating thrombotic disorders has been investigated over the years. Although administration of wild type human tPA has been the standard for treating acute myocardial infarction, a condition that kills more people worldwide than any other single disease, several factors have constrained its use as a therapeutic.
First, the activity of tPA is rapidly inhibited by the serpin plasminogen activator inhibitor type 1 (PAI-1) in vivo. Secondly, the enzyme is rapidly cleared from the circulation system and has a very short-half life. Third, platelet-rich thrombi are resistant to lysis by tPA and other fibrinolytic agents. To overcome some of these limiting factors of wild-type tPA and produce variants of tPA with better pharmacological profiles, studies of structure-function relationship of tPA have been carried out over the years. For example, domain deletion studies have implicated that kringle 2 (K2), and possibly the F domain, binds to fibrin and thereby mediate the fibrin-dependent activation of plasminogen by tPA. Deletion and mutation analysis also have demonstrated the role of structural elements located in the first three domains, i.e. F, EGF, and K1, in recognition by certain heptic receptors. Furthermore, it has been shown that the plasminogen activator inhibitor (PAI) binding site is located in the light chain at residues 296-304 of tPA. Thus, mutation in the binding site can empower the enzyme with resistance to PAI-1 (Madison et al., 1989. Nature 339, 721-724; U.S. Pat. No 5,550,042; 5,486,602).
PAI-1 is the primary inhibitor of tPA and other plasminogen activators in the blood. Under normal physiological conditions, PAI-1 limits the production of plasmin and serves to keep fibrinolysis in check. In certain pathological conditions, uncontrolled plasmin production can result in excessive degradation of fibrin and an increased risk of bleeding. During treatment of acute myocardial infarction, PAI-1 is a key culprit in diminishing the effectiveness of thrombolytic treatment by limiting the production of plasmin.
Recent attempts to improve tPA as a thrombolytic agent have focused primarily on substitution and/or deletion of some of the modules believed to be involved in tPA stability or its interaction with inhibitors such as PAI-1. Such efforts have culminated in the development of certain tPA-based therapeutics. For example, TNK-tPA is a derivative of tPA, in which a tetra-alanine substitution at amino acids 296-299 is introduced in the protease domain, thereby desensitizing the polypeptide to inhibition by PAI-1 (Paoni et al., 1993. Thromb Haemost. 70, 307-312; U.S. Pat. No. 5,246,850). TNK-tPA further contains mutations that abolish glycosylation in some sites to increase its half-life. In addition, an N-terminal truncated variant of tPA, rPA 06022, has been shown to prolong the half-life of the polypeptide (Kohnert et al., 1992. Protein Engineering 5, 93-100). Although second-generation tPA products, such as TNK-tPA (TNKase™, Tenecteplase) and rPA 06022 (Retavase®, reteplase), have overcome some of the problems set forth herein, these products, together with many other variants of tPA generated so far, do not solve an important problem: the resistance of platelet-rich thrombi to lysis, a phenomenon that often occurs in acute myocardial infarction (AMI) and other acute coronary syndromes (ACS).
In platelet-rich thrombi, the platelet aggregates are formed by the binding of either fibrinogen/fibrin or von Willebrand factor to the platelet membrane receptor, glycoprotein IIb/IIIa, the most abundant cell-surface protein in platelets. The blocking of the ligand binding function of glycoprotein IIb/IIIa has become one of the approaches used clinically to prevent platelet aggregation and thrombosis. As such, certain natural proteins, e.g. monoclonal antibodies, peptides, and small molecules against glycoprotein IIb/IIIa, have been identified that inhibit platelet aggregation by preventing the binding of fibrinogen/fibrin or von Willebrand factor to glycoprotein IIb/IIIa. One class of inhibitors includes the snake venom-derived disintegrins, a family of homologous peptides containing the (arginine-glycine-aspartate) RGD motif. These peptides have potent inhibitory effect on the binding of adhesive proteins to platelet glycoprotein IIb/IIIa, a process which is activated by many different stimuli (Ruoslahti E. and Pierschbacher M. D. 1987. Science 238, 491-497).
Fibrin binding to platelet glycoprotein IIb/IIIa receptors, followed by platelet-mediated clot retraction, creates a local area of high fibrin concentration, which limits the diffusion of fibrinolytic proteins such as tPA through the clot. Uncoupling fibrin from integrin receptors by inhibitor peptides can reduce the quantity of platelet-bound fibrin and accelerate the lysis of platelet-rich thrombus in a model system. This was further supported by clinical evidence showing that thrombolytic agents, when used in combination with abciximab (Reopro®), a therapeutic monoclonal antibody against glycoprotein IIb/IIIa that is capable of blocking platelet interactions with fibrinogen, can more effectively restore coronary flow in acute myocardial infarction.
In addition, studies have suggested that platelet activation and subsequent accumulation in microvessels are involved in the generation of infarcts and intracerebral hemorrhage (ICH), which sometimes can be caused by the administering of tPA to stroke patients. Studies using animal models have shown that platelet receptor glycoprotein IIb/IIIa inhibitor reduces the occurrence of tPA-induced intracerebral hemorrhage after thromboembolic stroke (Lapchak et al., 2002. Stroke, 33, 147-152).
Attempts have been made to generate integrin specific tPA. Smith et al. described the use of protein loop to construct variants of tPA that bind integrin receptor, in which a CDR3 region of an antibody against integrin αIIbβ3 was grafted into the EGF domain of tPA (Smith et al. 1995. J. Biol. Chem. 270: 30486-90). Yamada also attempted to tailor tPA mutants with affinity to integrin by inserting the RGD motif into either the Kringle I domain, the linker region between Kringle II and the protease domain, or the protease domain (Yamada et al., Bioch. Bioph. Res. Comm. 1996, 228, 306-331). According to Yamada, all of the tPA mutants generated (except 148RGD-tPA) were defective either in its catalytic activity or its ability to bind to integrin.
In light of the above, there remains a need in the art to provide methods and compositions useful for treating thrombotic disorders.